Each gold conjugate has a technical data sheet which indicates the following information: 1) Number of particles counted; 2) Mean particle diameter; 3) Coefficient of variation given as a percent; 4) Percent of single particles; 5) Percent of particles larger than triplets; and 6) Minimum detectable protein. The coefficient of variation is an important parameter in describing the relative distribution of gold particle sizes around the mean for a given batch. The coefficient of variation equals the standard deviation divided by the mean.
Normal Gaussian distributions work as follows: ±1 standard deviation describes 68% of the area under the curve; ± 2 standard deviations describe 95% of the area under the curve; ± 3 standard deviations describe 99.73% of the area under the curve. As an example, you have purchased a gold conjugate - Goat anti-Rabbit IgG (H+L), 10nm - having a mean particle diameter of 9.8nm with a coefficient of variation of 4.1%. First, the standard deviation needs to be determined. In this case it is 0.402nm (4.1% x mean particle diameter). Statistically, 68% of the particles will be from 9.40 to 10.20nm, 95% from 9.00 to 10.60nm and 99.73% from 8.60 to 11.00nm. A reliable size characterization has been determined for the batch.
All antigens and tissue react differently to different fixatives and preservation strategies (e.g. high pressure freezing, cryoultramicrotomy, etc.). As a result no one method will produce results all the time. Fixation is a very important step in determining the success of most immunolabeling procedures. Paraformaldehyde-based fixatives are most often used in concentrations from 0.5% up to 4% or higher. Often glutaraldehyde is added in concentrations typically from 0.1 to 0.5%. Cacodylate and the phosphate (Millonig, Sörensen) buffers are popular and the zwitterionic buffers such as HEPES or PIPES can be used as well for aldeyde fixation. The quenching of unreacted aldehydes is recommended by one of the following methods: 1) 0.05-0.1M ammonium chloride in buffer at 4°C for a few hours; 2) 0.1-0.2M glycine in buffer for a few hours; or 3) Sodium borohydride (0.1 mg/ml) in buffer for a few hours. Osmium tetroxide is not often used for tissues processed for immunolabeling. Berryman and Rodewald, 1990, J. Histochem, Cytochem., 38: 159-170 describe an excellent method to attain membrane contrast without the use of osmium.
The hydrophilic polar resins (Lowicryl K4M and K11M; L R White; L R Gold) have proven to be the best based on immunoreactivity. Cryoultramicrotomy methods are more difficult but yield excellent results when done correctly. The polar resins are partially water soluble and do not need to be dehydrated to 100%. Apolar hydrophobic resins (Lowicryl HM20 and HM23) are available for use as well. The epoxy resins, for most applications, are not recommended. Dehydration, infiltration and polymerization with polar and apolar resins can be done at room temperatures or in the cold (progressively lower temperature techniques). Polymerization of polar and apolar resins is done by UV for immuno applications; however, UV as well as oven cures can be done with LR White.
Positive as well as negative controls should be a part of all immunolabeling studies. Reagent and system dynamics require that controls be used to identify optimum signal-to-noise ratios (i.e. correct dilution of primary and secondary antibodies), viability of antibodies being used and necessary blocking steps or strategies. Tween 20® (Product 15713), Fish gelatin (45%) (Product15717) are good generic blocking reagents that can be mixed with antibody or rinse solutions.
Sample Labeling Protocol
Immunolabeling should be done in a humid chamber to protect against drying. The steps are:
1. Blocking step: 20-30 minutes at room temperature (recommend 0.1% Tween 20® and 0.1% Fish Gelatin to start. Add BSA 1-5% and the appropriate normal serum to reduce background if present).
2. Primary Antibody Incubation: 30-60 minutes at room temperature or 37°C.
3. Buffer Rinse: 30 minutes at room temperature with a buffer change every 5 minutes.
4. Gold Conjugate Incubation: 30-45 minutes in duration at room temperature or 37°C.
5. Buffer Rinse: A method found beneficial to reduce background is to elevate the NaCl concentration to approximately 2.5M from 1.54mM. The change in molarity has a beneficial effect on background due to ionic interaction of the gold conjugate (net negative change) and the substrate. Rinse for approximately 10 minutes and switch to water (ultrapure) for at least four changes of five minutes each.
6. Post Stain: 2% uranyl acetate (aqueous or alcoholic) for 5 minutes, rinse well in ultrapure water and then stain for 5 minutes with lead (25ml DI water, dissolve one sodium hydroxide pellet and add 0.125g of lead citrate). Rinse well after lead staining.
NOTE: Elevated temperatures (i.e. 37°C) during incubations require less time. Less time helps to keep the sections on the grids. Nickel or gold grids should be used. All grids, regardless, should be cleaned prior to use (1N HCl; 100% ETOH; 100% Acetone - Dry in 60°C oven on filter paper).
Questions and Customer Support: If there are any questions, please contact Ted Pella, Inc. and ask for Cindy Smith, Technical Product Specialist.